Recombinant yeast and substance production method using the same

ABSTRACT

Substance productivity is improved by introducing a metabolic pathway for synthesis of acetyl-CoA or acetic acid from glucose-6-phosphate into yeast. Acetic acid productivity, acetyl-CoA productivity, and productivity of a substance made from acetyl-CoA-derived are improved by attenuating genes involved in the glycolytic system of yeast and introducing a phosphoketolase gene into the yeast.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/979,537filed Jul. 12, 2013, which is U.S. National Stage of InternationalApplication no. PCT/JP2011/050974 filed Jan. 20, 2011. The entiredisclosures of the prior applications are considered part of thedisclosure of the accompanying continuation, and are hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to a recombinant yeast prepared throughmodification to suppress and/or enhance the expression of apredetermined gene or introduction of a predetermined gene, and asubstance production method using the recombinant yeast.

BACKGROUND ART

Examples of techniques concerning substance production using yeast aremainly methods for designing substance production pathways usingacetyl-CoA as an intermediate. For example, oleic acid, which is atypical fatty acid, requires 9 molecules of acetyl-CoA as a rawmaterial, and carotin, which is a typical diterpene, requires 12molecules of acetyl-CoA as a raw material. Accordingly, a technique forsynthesizing fatty acid useful as a pharmaceutical product or a finechemical (Patent Document 1), a technique for synthesizing terpenoid(Patent Document 2), and a technique for synthesizing polyketide (PatentDocument 3) using acetyl-CoA accumulated within yeast are known.Furthermore, examples of a substance that is synthesized usingacetyl-CoA as an intermediate include butanol (Patent Document 4),isopropanol (Patent Document 5) and farnesene (Patent Document 5), whichare attracting attention as biofuels.

In yeast, ethanol produced extracellularly is taken up by cells and thenacetyl-CoA is synthesized from the incorporated ethanol. When theconcentration of ethanol produced by yeast becomes high, the yeast's owngrowth is inhibited. Therefore, it has been difficult to increase theamount of acetyl-CoA within cells by means such as a means of increasingthe ethanol production capacity of yeast or a means of increasing theamount of ethanol to be taken up by yeast.

More specifically, Patent Document 2 discloses a technique forsynthesizing farnesene from acetyl-CoA, but the yield thereof is about25% of the theoretical yield. Moreover, Patent Document 6 discloses atechnique for synthesizing 6-methyl salicylate from acetyl-CoA, but theyield is about 20% of the theoretical yield. As described above,substance production from acetyl-CoA is problematic in that productivityis significantly low.

PRIOR PATENT DOCUMENTS

-   Patent Document 1: JP Patent Publication (Kokai) No. 63-287491 A    (1988)-   Patent Document 2: WO2008/045555-   Patent Document 3: JP Patent Publication (Kokai) No. 2008-22865 A-   Patent Document 4: WO2008/137406-   Patent Document 5: US2008/0293125-   Patent Document 6: US2006/0148052

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

In view of the above circumstances, an object of the present inventionis to provide a recombinant yeast with high substance productivity byintroducing a metabolic pathway for synthesis of acetyl-CoA or aceticacid from glucose-6-phosphate into yeast, in particular, and a substanceproduction method using the yeast.

Means for Solving Problem

As a result of intensive studies to achieve the above object, thepresent inventors have discovered that the productivity of acetic acid,acetyl-CoA, and a substance made from acetyl-CoA can be improved byattenuating a gene involved in the glycolytic system of yeast andintroducing a phosphoketolase gene into the yeast, and thus they havecompleted the present invention. In addition, the term “phosphoketolase”refers to an enzyme that catalyzes a reaction to convert xylulose5-phosphate into acetylphosphate and glyceraldehyde 3-phosphate.

Specifically, the present invention encompasses the following (1) to(7).

-   (1) A recombinant yeast, which comprises an attenuated    phosphofructokinase gene and an introduced phosphoketolase gene.-   (2) The recombinant yeast according to (1), wherein the expression    level of a glucose-6-phosphate dehydrogenase gene and/or a    D-ribulose-5-phosphate-3-epimerase gene is increased.-   (3) The recombinant yeast according to (1), wherein a    phosphotransacetylase gene is introduced and/or the expression level    of an acetyl-CoA synthetase gene is increased.-   (4) The recombinant yeast according to (1), wherein the expression    level of an alcohol acetyltransferase gene involved in a reaction    for synthesis of ethyl acetate using acetyl-CoA as a substrate is    increased.-   (5) The recombinant yeast according to (1), which is prepared by    introducing an acetoacetic acid decarboxylase gene, a    butyrate-acetoacetate CoA-transferase subunit A gene, a    butyrate-acetoacetate CoA-transferase subunit B gene, an acetyl-CoA    acetyltransferase gene, and an isopropanol dehydrogenase gene, which    are involved in a reaction for synthesis of isopropanol using    acetyl-CoA as a substrate.-   (6) A method for producing a substance, comprising a step of    culturing the recombinant yeast of any one of (1) to (5) above in    medium.-   (7) The method for producing a material according to (6), wherein    the substance is 1 type of substance selected from the group    consisting of acetic acid, acetyl-CoA, ethyl acetate made from    acetyl-CoA, and isopropanol made from acetyl-CoA.

Effects of the Invention

The recombinant yeast according to the present invention has attenuatedactivity of converting fructose-6-phosphate intofructose-1,6-bisphosphate, and, imparted activity of converting xylulose5-phosphate into acetylphosphate. Accordingly, through the use of therecombinant yeast according to the present invention, the productivityof acetic acid or a substance made from acetyl-CoA can be improved.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic diagram showing a part of a glycolytic systemincluding a metabolic pathway in which a phosphofructokinase gene to beattenuated in the yeast according to the present invention is involved.

FIG. 2 is a characteristic diagram showing a part of a pentose phosphatesystem including a metabolic pathway in which a phosphoketolase gene tobe introduced into the yeast according to the present invention isinvolved.

FIG. 3 is a characteristic diagram showing the result of conductingmolecular phylogenetic tree analysis of phosphoketolase genes derivedfrom various organisms.

FIG. 4 is a characteristic diagram showing a pathway for synthesis ofacetyl-CoA from acetylphosphate and acetic acid and a pathway forsynthesis of another substance from acetyl-CoA.

FIG. 5 is a characteristic diagram showing the result of conductingmolecular phylogenetic tree analysis of phosphotransacetylase genesderived from various organisms.

FIG. 6 is a flow chart for construction of a pESC-HIS-ZWF1-RPE1 vector.

FIG. 7 is a flow chart for construction of a pESC-Leu-PKT vector and apESC-Leu-PKT-PTA vector.

FIG. 8 is a flow chart for construction of a pESC-Trp-ATF1 vector.

FIG. 9 is a flow chart for construction of a vector for disruption of aPFK1 gene.

FIG. 10 is a flow chart for construction of a vector for disruption of aPFK2 gene.

FIG. 11 is a characteristic diagram showing the results of an aceticacid production test.

FIG. 12 is a characteristic diagram showing the results of an ethylacetate production test.

FIG. 13 is a characteristic diagram showing the results of anisopropanol production test.

FIG. 14 is a characteristic diagram showing the results of conductingmetabolome analysis of yeast by CE-TOFMS.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

The present invention will be described in detail as follows withreference to drawings and examples.

The recombinant yeast according to the present invention comprises anattenuated gene that encodes an enzyme involved in a glycolytic system,and an introduced phosphoketolase gene. The recombinant yeast hasactivity to convert xylulose 5-phosphate to acetylphosphate. Examples ofyeast that can be used as a host include, but are not particularlylimited to, yeast of the genus Candida such as Candida Shehatae, yeastof the genus Pichia such as Pichia stipitis, yeast of the genusPachysolen such as Pachysolen tannophilus, yeast of the genusSaccharomyces such as Saccharomyces cerevisiae, and yeast of the genusSchizosaccharomyces such as Schizosaccharomyces pombe. In particular,Saccharomyces cerevisiae is preferred. A yeast strain to be used hereinmay be an experimental strain to be used for convenience of experimentsor an industrial strain (practical strain) to be employed for practicalusefulness. Examples of such an industrial strain include yeast strainsto be used for production of wine, sake, and shochu (spirits).

Here, an example of a gene that encodes an enzyme involved in aglycolytic system and is subjected to attenuation is aphosphofructokinase gene.

Furthermore, as an enzyme involved in the glycolytic system, hexokinase,glucose phosphate isomerase, aldolase, triosephosphate isomerase,glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase,phosphoglyceromutase, enolase, and pyruvate kinase are known in additionto phosphofructokinase. Genes encoding these enzymes other thanphosphofructokinase may also be attenuated.

The phosphofructokinase gene encodes an enzyme that convertsfructose-6-phosphate into fructose-1,6-bisphosphate in the glycolyticsystem, as shown in FIG. 1. The expression, “the phosphofructokinasegene is attenuated” means that phosphofructokinase activity issignificantly lowered. In other words, the expression means that theamount of fructose-1,6-bisphosphate to be synthesized through theglycolytic system is significantly decreased. Examples of means forattenuating a phosphofructokinase gene include, but are not particularlylimited to, disruption or deletion of the phosphofructokinase gene,disruption or deletion of the expression control region of thephosphofructokinase gene, addition of an inhibitor (e.g., citric acid)of phosphofructokinase, and a technique for suppressing the expressionof the phosphofructokinase gene with the use of a method using RNAinterference such as siRNA or an antisense method.

In addition, as endogenous phosphofructokinase genes of Saccharomycescerevisiae, a PFK1 gene and a PFK2 gene are known (THE JOURNAL OFBIOLOGICAL CHEMISTRY, Vol. 275, No. 52, Issue of December 29, pp.40952-40960, 2000). When Saccharomyces cerevisiae is used as a host forthe recombinant yeast according to the present invention, either thePFK1 gene or the PFK2 gene may be attenuated or both genes may beattenuated. Moreover, endogenous phosphofructokinase genes of yeastother than Saccharomyces cerevisiae are also known and can be specifiedreferring to existing databases such as Genbank, DDBJ, and EMBL. Asdescribed above, phosphofructokinase genes specified by the abovetechniques and/or means can be attenuated by specifying endogenousphosphofructokinase genes of various types of yeast.

Furthermore, the recombinant yeast according to the present inventionacquires the capacity to convert xylulose 5-phosphate intoacetylphosphate through exogenous introduction of a phosphoketolase gene(PKT gene). In addition, xylulose 5-phosphate is synthesized as ametabolite resulting from yeast's original pentose phosphate system fromribulose-5-phosphate (FIG. 2). Acetylphosphate synthesized byphosphoketolase is converted into acetic acid by yeast's original aceticacid kinase. Therefore, in the recombinant yeast prepared by attenuatinga phosphofructokinase gene and introducing a phosphoketolase gene, theproductivity of acetic acid to be secreted to medium is drasticallyimproved.

Examples of phosphoketolase genes to be preferably used herein include,but are not particularly limited to, phosphoketolase genes derived fromlactic acid bacteria or bifidobacteria having a metabolic pathway forheterolactic fermentation. Here, the term “heterolactic fermentation”refers to fermentation whereby pyruvic acid generated via the glycolyticsystem from glucose is metabolized to give not only lactic acid, butalso ethanol, acetic acid, and carbon dioxide. Through such heterolacticfermentation, ethanol or acetic acid is synthesized from acetylphosphatethat is generated by phosphoketolase.

More specifically, as phosphoketolase genes, as shown in FIG. 3,phosphoketolase genes derived from various microorganisms can be used.In addition, Table 1 below shows the relationship between numbersassigned in the molecular phylogenetic tree shown in FIG. 3 andmicroorganisms.

TABLE 1 1 Bifidobacterium_animalis 2 Bifidobacterium_longum 3Bifidobacterium_adolescentis 4 Bifidobacterium_pullorum 5Lactobacillus_plantarum_1 6 Lactobacillus_plantarum_2 7Lactobacillus_pentosus 8 Lactobacillus_sakei 9 Lactobacillus_salivarius10 Lactobacillus_reuteri 11 Lactobacillus_johnsonii 12Lactobacillus_casei 13 Bacillus_coagulans 14 Leuconostoc_mesenteroides_115 Leuconostoc_mesenteroides_2 16 Streptococcus_gordonii 17Clostridium_acetobutylicum 18 Clostridium_butyricum 19Mycoplasma_agalactiae 20 Cellulomonas_flavigena 21Methylobacterium_populi 22 Mycobacterium_gilvum 23Mycobacterium_vanbaalenii 24 Nitrosococcus_oceani 25 Synechococcus_sp.26 Rhizobium_leguminosarum 27 Nocardioides_sp. 28 Anabaena_variabilis 29Bacteroides_capillosus 30 Marinobacter_aquaeolei 31 Acidovorax_sp. 32Pseudamonas_aeruginosa 33 Maricaulis_maris 34 Cyanothece_sp. 35Mariprofundus_ferrooxydans 36 Nostoc_punctiforme 37 Aspergullus_oryzae_138 Aspergullus_oryzae_2 39 Aspergullus_nidulans 40Aspergillus_fumigatus_1 41 Aspergillus_fumigatus_2 42Cryptococcus_neoformans_1 43 Cryptococcus_neoformans_2 44Penicillium_chrysogenum 45 Neosartorya_fischeri_1 46Neosartorya_fischeri_2 47 Aspergillus_niger_1 48 Aspergillus_niger_2 49Aspergillus_terreus 50 Aspergillus_clavatus 51 Sclerotinia_sclerotiorum52 Botryotinia_fuckeliana 53 Phaeosphaeria_nodorum 54Pyrenophora_tritici-repentis 55 Neurospora_crassa_1 56Neurospora_crassa_2 57 Podospora_anserina 58 Magnaporthe_grisea 59Ustilago_maydis

As phosphoketolase genes, phosphoketolase genes classified within thebroken-line frame of the molecular phylogenetic tree shown in FIG. 3 arepreferably used. Phosphoketolase genes classified in the group aremainly derived from lactic acid bacteria or bifidobacteria havingmetabolic pathways for heterolactic fermentation. Further specifically,as phosphoketolase genes classified within the broken-line frame of themolecular phylogenetic tree shown in FIG. 3, genes derived frommicroorganisms belonging to the genus Bifidobacterium that arebifidobacteria, microorganisms belonging to the genus Lactobacillus thatare lactic acid bacteria, or microorganisms belonging to the genusLeuconostoc are preferably used. Further specifically, as aphosphoketolase gene classified within the broken-line frame of themolecular phylogenetic tree shown in FIG. 3, a gene encodingphosphoketolase comprising the amino acid sequence shown in any one ofSEQ ID NOS: 1 to 19 can be used. The amino acid sequence of SEQ ID NO: 1is the amino acid sequence encoded by a Bifidobacterium animalis-derivedphosphoketolase gene. The amino acid sequences shown in SEQ ID NOS: 2 to19 are the amino acid sequences encoded by phosphoketolase genes derivedfrom Bifidobacterium longum, Bifidobacterium adolescentis,Bifidobacterium pullorum, Lactobacillus plantarum, Lactobacillusplantarum, Lactobacillus pentosus, Lactobacillus sakei, Lactobacillussalivarius, Lactobacillus reuteri, Lactobacillus johnsonii,Lactobacillus casei, Bacillus coagulans, Leuconostoc mesenteroides,Leuconostoc mesenteroides, Streptococcus gordonii, Clostridiumacetobutylicum, Clostridium butyricum, and Mycoplasma agalactiae,respectively.

Specifically, in the present invention, a phosphoketolase gene encodingthe amino acid sequence of any one of SEQ ID NOS: 1 to 19 is preferablyused. In particular, as phosphoketolase genes, Bifidobacterium animalis(SEQ ID NO: 1), Bifidobacterium longum (SEQ ID NO: 2), Bifidobacteriumadolescentis (SEQ ID NO: 3), and Bifidobacterium pullorum (SEQ ID NO: 4)are most preferably used.

Furthermore, a phosphoketolase gene may consist of a polynucleotideencoding a protein that consists of an amino acid sequence having adeletion, a substitution, an addition, or an insertion of 1 or severalamino acids with respect to the amino acid sequence of any one of SEQ IDNOS: 1 to 19 and has phosphoketolase activity. Here, the term “severalamino acids” refers to, for example, 2 to 100, preferably 2 to 80, morepreferably 2 to 55, and further preferably 2 to 15 amino acids.

Furthermore, a phosphoketolase gene may consist of a polynucleotideencoding a protein that consists of an amino acid sequence having 80% ormore, preferably 85% or more, more preferably 90% or more, and furtherpreferably 98% or more sequence similarity with respect to the aminoacid sequence of any one of SEQ ID NOS: 1 to 19, and has phosphoketolaseactivity. Here, the term “sequence similarity” refers to a value that iscalculated to represent similarlity between two amino acid sequenceswhen sequence similarity search software such as BLAST, PSI-BLAST, orHMMER is used with default settings.

Here, the term “phosphoketolase activity” refers to activity to convertxylulose-5-phosphate to acetylphosphate. Therefore, whether or not apredetermined protein has phosphoketolase activity can be determinedbased on the amount of acetylphosphate synthesized, using a reactionsolution containing xylulose-5-phosphate as a substrate (e.g., JOURNALOF BACTERIOLOGY, Vol. 183, No. 9, May 2001, p. 2929-2936).

The recombinant yeast according to the present invention can increasethe amount of acetylphosphate synthesized because of the presence of aphosphoketolase gene introduced, by enhancing the expression of anenzyme gene involved in the pentose phosphate system shown in FIG. 2. Asa result, it can increase the amount of synthesized acetic acid.Examples of genes to be subjected to enhancement of expression in thepentose phosphate system shown in FIG. 2 include, but are notparticularly limited to, a glucose-6-phosphate dehydrogenase gene and aribulose-5-phosphate-3-epimerase gene. Moreover, the expression ofeither one of or both of these genes may be enhanced.

Furthermore, an endogenous glucose-6-phosphate dehydrogenase gene ofSaccharomyces cerevisiae is known as a ZWF1 gene. Also, an endogenousribulose-5-phosphate-3-epimerase gene of Saccharomyces cerevisiae isknown as an RPE1 gene. Endogenous glucose-6-phosphate dehydrogenasegenes or ribulose-5-phosphate-3-epimerase genes are known for yeastother than Saccharomyces cerevisiae and can be specified referring tothe existing databases such as Genbank, DDBJ, and EMBL.

Here, the expression “gene expression is enhanced” refers to significantimprovement in activity of an enzyme to be encoded by a subject gene,and is meant to include a significant increase in the expression levelof such a gene. An example of a technique for enhancing gene expressionis a technique for significantly increasing the expression level of therelevant gene. Examples of a technique for increasing the expressionlevel of a specific gene include, but are not particularly limited to, atechnique that involves modifying the expression control region of anendogenous gene of a chromosome and a technique that involvesintroducing a vector having the relevant gene located downstream of apromoter with high activity.

Meanwhile, the recombinant yeast according to the present invention canincrease the amount of acetyl-CoA synthesized by further introducing aphosphotransacetylase gene (PTA gene) or further enhancing an acetyl-CoAsynthetase gene (ACS gene) as shown in FIG. 4, in addition toattenuation of a phosphofructokinase gene and introduction of aphosphoketolase gene.

A phosphotransacetylase gene is not yeast's original gene and thus isintroduced as a foreign gene. Examples of such a phosphotransacetylasegene are not particularly limited, and genes referred to as PTA genes invarious bacteria are broadly applicable.

More specifically, as phosphotransacetylase genes, as shown in FIG. 5,phosphotransacetylase genes derived from various microorganisms can beused. In addition, Table 2 below shows the relationship between numbersassigned in the molecular phylogenetic tree in FIG. 5 andmicroorganisms.

TABLE 2 1 Bacillus_subtllis 2 Bacillus_amyloliquefaciens 3Bacillus_licheniformis 4 Geobacillus_thermodenitrificans 5Listeria_innocua 6 Staphylococcus_aureus 7 Lactococcus_lactis 8Carnobacterium_sp. 9 Mycobacterium_vanbaalenii 10Clostridium_perfringens 11 Enterococcus_faecalis 12Leuconostoc_mesenteroides 13 Clostridium_acetobutylicum 14Bifidobacterium_animalis_lactis 15 Corynebacterium_glutamicum 16Escherichia_coli_K-12 17 Escherichia_coli_53638 18 Vibrio_vulnificus 19Haemophilus somnus 20 Yersinia_pestis 21 Shigella_sonnei

The origins of the following PTA genes are shown in FIG. 5 and Table 2.The amino acid sequence of a protein encoded by the Bacillussubtilis-derived PTA gene is shown in SEQ ID NO: 20, the amino acidsequence of a protein encoded by the Bacillus amyloliquefaciens-derivedPTA gene is shown in SEQ ID NO: 21, the amino acid sequence of a proteinencoded by the Bacillus licheniformis-derived PTA gene is shown in SEQID NO: 22, the amino acid sequence of a protein encoded by theGeobacillus thermodenitrificans-derived PTA gene is shown in SEQ ID NO:23, the amino acid sequence of a protein encoded by the Listeriainnocua-derived PTA gene is shown in SEQ ID NO: 24, the amino acidsequence of a protein encoded by the Staphylococcus aureus-derived PTAgene is shown in SEQ ID NO: 25, the amino acid sequence of a proteinencoded by the Lactococcus lactis-derived PTA gene is shown in SEQ IDNO: 26, the amino acid sequence of a protein encoded by theCarnobacterium sp.-derived PTA gene is shown in SEQ ID NO: 27, the aminoacid sequence of a protein encoded by the Mycobacteriumvanbaalenii-derived PTA gene is shown in SEQ ID NO: 28, the amino acidsequence of a protein encoded by the Clostridium perfringens-derived PTAgene is shown in SEQ ID NO: 29, the amino acid sequence of a proteinencoded by the Enterococcus faecalis-derived PTA gene is shown in SEQ IDNO: 30, the amino acid sequence of a protein encoded by the Leuconostocmesenteroides-derived PTA gene is shown in SEQ ID NO: 31, the amino acidsequence of a protein encoded by the Clostridium acetobutylicum-derivedPTA gene is shown in SEQ ID NO: 32, the amino acid sequence of a proteinencoded by the Bifidobacterium animalis_lactis-derived PTA gene is shownin SEQ ID NO: 33, the amino acid sequence of a protein encoded by theCorynebacterium glutamicum-derived PTA gene is shown in SEQ ID NO: 34,the amino acid sequence of a protein encoded by the Escherichia coliK-12-derived PTA gene is shown in SEQ ID NO: 35, the amino acid sequenceof a protein encoded by the Escherichia coli 53638-derived PTA gene isshown in SEQ ID NO: 36, the amino acid sequence of a protein encoded bythe Vibrio vulnificus-derived PTA gene is shown in SEQ ID NO: 37, theamino acid sequence of a protein encoded by the Haemophilussomnus-derived PTA gene is shown in SEQ ID NO: 38, the amino acidsequence of a protein encoded by the Yersinia pestis-derived PTA gene isshown in SEQ ID NO: 39, and the amino acid sequence of a protein encodedby the Shigella sonnei-derived PTA gene is shown in SEQ ID NO: 40.

In addition, the phosphotransacetylase gene may consist of apolynucleotide encoding a protein that consists of an amino acidsequence having a deletion, a substitution, an addition, or an insertionof 1 or several amino acids with respect to the amino acid sequence ofany one of SEQ ID NOS: 20 to 40, and has phosphotransacetylase activity.Here, the term “several amino acids” refers to, for example, 2 to 35,preferably 2 to 25, more preferably 2 to 15, and further preferably 2 to10 amino acids.

Furthermore, the phosphotransacetylase gene may consist of apolynucleotide encoding a protein that consists of an amino acidsequence having 80% or more, preferably 85% or more, more preferably 90%or more, and further preferably 98% or more sequence similarity withrespect to the amino acid sequence of any one of SEQ ID NOS: 20 to 40,and has phosphotransacetylase activity. Here, the term “sequencesimilarity” refers to a value that is calculated to representsimilarlity between two amino acid sequences when sequence similaritysearch software such as BLAST, PSI-BLAST, or HMMER is used with defaultsettings.

Here, the term “phosphotransacetylase activity” refers to activity totransfer CoA to acetylphosphate. Therefore, whether or not apredetermined protein has phosphotransacetylase activity can bedetermined based on the amount of acetyl-CoA synthesized using areaction solution containing acetylphosphate and CoA.

Furthermore, the acetyl-CoA synthetase gene shown in FIG. 4 is yeast'soriginal gene. Hence, for enhancement of such an acetyl-CoA synthetasegene, a technique that involves modifying the expression control regionof the relevant endogenous gene of a chromosome and a technique thatinvolves introducing a vector having the relevant gene locateddownstream of a promoter having high activity are applicable. Inaddition, as endogenous acetyl-CoA synthetase genes of Saccharomycescerevisiae, an ACS 1 gene and an ACS2 gene are known. Endogenousacetyl-CoA synthetase genes of yeast other than Saccharomyces cerevisiaeare also known and can be specified referring to existing databases suchas Genbank, DDBJ, and EMBL.

As described above, in the recombinant yeast according to the presentinvention, the amount of acetylphosphate synthesized; that is, theamount of acetic acid synthesized is significantly increased (FIG. 2) orthe amount of acetyl-CoA synthesized is significantly increased (FIG.4). Therefore, the recombinant yeast according to the present inventioncan be used when acetic acid or acetyl-CoA is a substance to beproduced. Alternatively, the recombinant microorganism according to thepresent invention can be used as a host for further modification toenable production of another substance (in FIG. 4, denoted as asubstance made from acetyl-CoA) using acetyl-CoA as a substrate.

Specifically, examples of such a substance made from acetyl-CoA that canbe synthesized include, but are not particularly limited to, butanol,alkane, propanol, fatty acid, fatty acid ester, acetone, acetoaceticacid, ethyl acetate, polyketide, amino acid, and terpenoid. When theseare substances to be produced, the productivity thereof can besignificantly improved using the recombinant yeast according to thepresent invention.

When isopropanol is a substance to be produced, for example, withreference to APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2007, p.7814-7818, Vol. 73, No. 24, a gene to be further introduced into therecombinant microorganism according to the present invention can bespecified. Furthermore, when polyketide is a substance to be produced,with reference to Proc. Natl. Acad. Sci. U.S.A., Vol. 95, pp. 505-509,January 1998, a gene to be further introduced into the recombinantmicroorganism according to the present invention can be specified.Moreover, when fatty acid is a substance to be produced, for example,with reference to Eur. J. Biochem. 112, p. 431-442 (1980) orMICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, September 2004, p. 501-517,a gene (e.g., a FAS gene) to be further introduced to or enhanced in therecombinant microorganism according to the present invention can bespecified. Furthermore, when alkane is a substance to be produced, agene that is involved in aldehyde synthesis from fatty acid and furtheralkane synthesis from aldehyde and should be further introduced into therecombinant microorganism according to the present invention can bespecified with reference to Science vol. 329 30 July pp. 559-562, forexample.

Furthermore, the expression of an endogenous alcohol acetyltransferasegene (ATF1 gene) of yeast is further enhanced, so that the amount ofethyl acetate synthesized from acetyl-CoA can be increased.Specifically, when ethyl acetate is a substance to be produced, theexpression of such an endogenous ATF1 gene is preferably enhanced.

Also, as described above, when the expression of a predetermined gene isenhanced, an appropriate promoter with high transcriptional activity isused. Examples of such a promoter that can be used herein include, butare not particularly limited to, a glyceraldehyde-3-phosphatedehydrogenase gene (TDH3) promoter, a 3-phosphoglyceratekinase gene(PGK1) promoter, and a high osmotic pressure-responsive 7 gene (HOR7)promoter. Of these, a pyruvate decarboxylase gene (PDC1) promoter ispreferred because of its high capacity to cause high-level expression ofa gene of interest located downstream thereof. Furthermore, through theuse of a gall promoter, a gal10 promoter, a heat shock protein promoter,a MFα1 promoter, a PHOS promoter, a GAP promoter, an ADH promoter, anAOX1 promoter, or the like, a gene downstream thereof can be stronglyexpressed.

Also, as a method for introducing the above gene, any conventionallyknown technique that is known as a method for yeast transformation isapplicable. Specifically, for example, gene introduction can beperformed by a method described in an electroporation method “Meth.Enzym., 194, p 182 (1990),” a spheroplast method “Proc. Natl. Acad. Sci.U.S.A., 75 p 1929 (1978),” a lithium acetate method “J. Bacteriology,153, p 163 (1983),” Proc. Natl. Acad. Sci. U.S.A., 75 p 1929 (1978),Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor LaboratoryCourse Manual, or the like. Examples thereof are not limited to thesemethods.

When a substance is produced using the above-explained recombinantyeast, the yeast is cultured in a medium containing an appropriatecarbon source. More specifically, recombinant yeast pre-culturedaccording to a conventional method is cultured in a medium so as tocause it to produce a substance of interest. For example, when butanol,alkane, propanol, fatty acid, fatty acid ester, acetone, acetoaceticacid, acetic ester, polyketide, amino acid, and terpenoid are producedas substances of interest, these substances of interest are synthesizedin a medium. Hence, after cells are separated from the medium by a meanssuch as centrifugation, such substances can be isolated from thesupernatant fraction. To isolate such substances from a supernatantfraction, for example, an organic solvent such as ethyl acetate ormethanol is added to the supernatant fraction, and then the resultant issufficiently stirred. An aqueous layer is separated from a solventlater, and then the substances can be extracted from the solvent layer.

EXAMPLES

The present invention is hereafter described in greater detail withreference to the following examples, although the technical scope of thepresent invention is not limited thereto.

In these examples, recombinant yeast was prepared by attenuating theendogenous phosphofructokinase gene of wild-type yeast orisopropanol-producing yeast and introducing a phosphoketolase gene intothe yeast. Recombinant yeast was further prepared by introducing orenhancing other genes in addition to the aforementioned gene attenuationand gene introduction. These strains were then examined for acetic acidproductivity, ethyl acetate productivity, and isopropanol productivity.

[Materials and Methods] Hosts

ECOS Competent E. coli JM109 (Nippon Gene Co., Ltd.), S. cerevisiaeYPH499 (Stratagene) as wild-type yeast, and a #15-10 strain (disclosedin the reference example described later) as isopropanol-producing yeastwere used.

Plasmid

<Preparation of pESCpgkgap-HIS>

PCR was performed under the following conditions. (Primers)

EcoRI-Pgap-F: (SEQ ID NO: 41) 5′-CACGGAATTCCAGTTCGAGTTTATCATTATCAA-3′BamHI-Pgap-R: (SEQ ID NO: 42) 5′-CTCTGGATCCTTTGTTTGTTTATGTGTGTTTATTC-3′

(PCR Conditions)

-   Template: 1 ng of pDI626 plasmid (see JP Patent Publication (Kokai)    No. 2005-52046 A)-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (2 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (2 minutes))×25 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

After PCR under the above conditions, a PCR product contained in thereaction solution was purified using a MinElute PCR purification kit(QIAGEN). Subsequently, the PCR product was digested with restrictionenzymes Barn HI and EcoR I. Agarose gel electrophoresis was performed, a686-bp fragment was excised, and the fragment was thus purified using aMiniElute Gel extraction kit (QIAGEN). Furthermore, the resultant wasligated to a pESC-HIS vector digested with restriction enzymes Barn HIand EcoR I. The thus obtained plasmid was designated as pESCgap-HIS.

Next, PCR was performed under the following conditions.

(Primers)

MunI-Ppgk1-F: (SEQ ID NO: 43) 5′-TAGGCAATTGCAAGAATTACTCGTGAGTAAGG-3′EcoRI-Ppgk1-R: (SEQ ID NO: 44)5′-ATAAGAATTCTGTTTTATATTTGTTGTAAAAAGTAG-3′

(PCR Conditions)

-   Template: pDI626PGK plasmid 1 ng-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (2 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (2 minutes))×25 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

After PCR under the above conditions, a PCR product contained in thereaction solution was purified using a MinElute PCR purification kit(QIAGEN). Subsequently, the PCR product was digested with restrictionenzymes Mun I and EcoR I. Agarose gel electrophoresis was performed, a718-bp fragment was excised, and the fragment was thus purified using aMiniElute Gel extraction kit (QIAGEN). Furthermore, the resultant wasligated to a pESCgap-HIS vector digested with a restriction enzyme EcoRI and then subjected to BAP treatment. The thus obtained plasmid wasdesignated as pESCpgkgap-HIS.

<Construction of pESCpgkgap-LEU>

After digestion of the above pESCpgkgap-HIS with restriction enzymesBarn HI and Not I, a 1427-bp fragment was excised and ligated to apESC-LEU vector (Stratagene) digested with restriction enzymes Barn HIand Not I in a similar manner.

<Construction of pESCpgkgap-TRP>

After digestion of the above pESCpgkgap-HIS with restriction enzymesBarn HI and Not I, a 1427-bp fragment was excised and then ligated to apESC-TRP vector (Stratagene) digested with restriction enzymes Barn HIand Not I in a similar manner.

<Construction of pESCpgkgap-URA>

After digestion of the above pESCpgkgap-HIS with restriction enzymesBarn HI and Not I, a 1427-bp fragment was excised and then ligated to apESC-URA vector (Stratagene) digested with restriction enzymes Barn HIand Not I in a similar manner.

<Construction of Other Vectors>

Upon construction of a pESC-HIS-ZWF1-RPE1 vector for enhancing theexpression of a ZWF1 gene and a RPE1 gene, a pESC-Leu-PKT vector forintroducing a PKT gene, a pESC-Leu-PKT-PTA vector for introducing thePKT gene and a PTA gene, a pESC-Trp-ATF1 vector for enhancing theexpression of an ATF1 gene, and a vector for disrupting a PFK1 gene anda PFK2 gene, PCR was performed with the following composition under thefollowing conditions. In addition, KOD-Plus-Ver.2 (TOYOBO) was used asDNA polymerase.

TABLE 3 <Composition> 10× Buffer 5 ul 2.5 mM dNTP 5 ul 25 mM MgSO₄ 4 ulfwd primer 1.5 ul  rev primer 1.5 ul  Genome (100 ng/ul) 1 ul DNApolymerase 1 ul H₂O 31 ul 

TABLE 4 <PCR conditions> 94 degrees C., 2 min −> 98 degrees C. 10 sec,Tm-5 degrees C. 30 sec, 68 degrees C. 5 min (×30) −>68 degrees C. 5 min

FIG. 6 shows a flow chart for construction of the pESC-HIS-ZWF1-RPE1vector. FIG. 7 shows a flow chart for construction of the pESC-Leu-PKTvector and the pESC-Leu-PKT-PTA vector. FIG. 8 shows a flow chart forconstruction of the pESC-Trp-ATF1 vector. FIG. 9 shows a flow chart forconstruction of the vector for disrupting the PFK1 gene. FIG. 10 shows aflow chart for construction of the vector for disrupting the PFK2 gene.Primers used in the flow charts for vector construction shown in FIGS.6-10 are listed in Table 5.

TABLE 5 Gene name Primer Nucleotide sequence ZWF1 G6PD-fwdcgcggatccgcggggcccATAAGGCAAGATGAGTGAAGGCCCC SEQ ID NO: 45 G6PD-revccgctcgagcgggtcgacGTGCTTGCATTTTTCTAATTATCCT SEQ ID NO: 46 RPE1 RPE1-fwdggaattccgcggccgcAGGTAAACACACAAGAAAAAATGG SEQ ID NO: 47 RPE1-revccttaattaagggactagtcTAAGAAATGCCGCATATGTAC SEQ ID NO: 48 PFK1 PFK1-fwdtcccccgggggacgagctcgCTCAGTTTCTTCTTGAAATTTAGCATCGTG SEQ ID NO: 49PFK1-rev tcccccgggggacgagctcgAAACGGAAAGAAAAAAGGCCGAC SEQ ID NO: 50PFK1-Inf-fwd CTAGAGGATCCCCGGGTACCCTCAGTTTCTTCTTGAAATTTAGCATCGTGSEQ ID NO: 51 PFK1-Inf-rvs CGGCCAGTGAATTCGAGCTCAAACGGAAAGAAAAAAGGCCGACSEQ ID NO: 52 PFK2 PFK2-fwdtcccccgggggacgagctcgTCCGGTCTTTATCTACCATTCATTTATTAC SEQ ID NO: 53PFK2-rev tcccccgggggacgagctcgGGTTTCATGGGGTAGTACTTGTATTA SEQ ID NO: 54PFK2-Inf-fwd CTAGAGGATCCCCGGGTACCTCCGGTCTTTATCTACCATTCATTTATTACSEQ ID NO: 55 PFK2-Inf-rvsCGGCCAGTGAATTCGAGCTCGGTTTCATGGGGTAGTACTTGTATTA SEQ ID NO: 56 ATF1ATF1-Inf-fwd tacgactcactatagggcccTTTGGCCTGGTATTGTCATC SEQ ID NO: 57ATF1-Inf-rev tctgttccatgtcgacCGACGATTCTGACCCTTTC SEQ ID NO: 58 ACS1ACS1-Inf-fwd GGGCCCGGGCGTCGACAGCAAAACCAAACATATCAA SEQ ID NO: 59ACS1-Inf-rev ACCAAGCTTACTCGAGCACACGAAAAAAAAAAAGTCG SEQ ID NO: 60 ACS2ACS2-Inf-fwd GGGCCCGGGCGTCGACTGTTATACACAAACAGAATA SEQ ID NO: 61ACS2-Inf-rev ACCAAGCTTACTCGAGAGAAAAGGAGCGAAATTTTATC SEQ ID NO: 62

In addition, PCR products were purified using a QIAquick PCRPurification Kit (QIAGEN). Furthermore, in the flow charts for vectorconstruction as shown in FIGS. 6 to 10, a Zero Blunt TOPO PCR cloningkit (Invitrogen) was used for TOPO cloning of PCR products. Also,pAUR135 was purchased from Takara Bio Inc. In the flow charts for vectorconstruction as shown in FIGS. 6 to 10, DNA fragments were excised fromagarose gel using a QIAquick Gel Extraction Kit (QIAGEN). In the flowcharts for vector construction as shown in FIGS. 6 to 10, vectors wereligated to DNA fragments by ligation reaction using aLigation-convenience Kit (Nippon Gene Co., Ltd.) or in-fusion reactionusing an In-Fusion Dry-Down PCR Cloning Kit (Clontech).

Furthermore, in the flow chart for vector construction as shown in FIG.7, vectors denoted as pBSK-PKT and pBSK-PTA were constructed bysynthesizing 5 types of PKT gene and 3 types of PTA gene, respectively,and then inserting them to the Sma I site of pBluescript IISK(+). Inaddition, these 5 types of PKT gene and 3 types of PTA gene were alloptimized for codons for Saccharomyces cerevisiae.

The 5 types of PKT gene were Bifidobacterium animalis subsp.lactis-derived phosphoketolase genes (SEQ ID NOS: 63 and 64),Aspergillus oryzae RIB40-derived phosphoketolase I genes (SEQ ID NOS: 65and 66), Aspergillus oryzae RIB40-derived phosphoketolase II genes (SEQID NOS: 67 and 68), Nostoc punctiforme ATCC 29133-derivedphosphoketolase genes (SEQ ID NOS: 69 and 70), and Marinobacteraquaeolei ATCC 700491-derived phosphoketolase genes (SEQ ID NOS: 71 and72). Furthermore, the 3 types of PTA gene were Bacillus subtilis subsp.subtilis str.168-derived phosphate acetyltransferase genes (SEQ ID NOS:73 and 74), Bifidobacterium animalis subsp. lactis AD011-derivedphosphate acetyltransferase genes (SEQ ID NOS: 75 and 76), andEscherichia coli K-12 MG1655-derived phosphate acetyltransferase genes(SEQ ID NOS: 77 and 78)

Transformation

Escherichia coli was transformed according to the protocols includedwith ECOS Competent E. coli JM109 (Nippon Gene Co., Ltd.). Yeast wastransformed according to the protocols included with a Frozen-EZ YeastTransformation II Kit (Zymo Research). Yeast gene disruption using thevector for disrupting the PFK1 gene and the PFK2 gene was performedaccording to the protocols included with pAUR135DNA (Takara Bio Inc.).

Acetic Acid Production Test

A transformant was cultured as follows. After active colony formation inan SD-His, Leu agar medium, cells were inoculated to 2 ml of an SD-His,Leu medium in a 15-ml test tube and then cultured overnight at 30degrees C. (Oriental Giken Inc. IFM, 130 rpm). The thus pre-culturedsolution was inoculated to 100 ml of an SD-His, Leu medium in a 500-mlErlenmeyer flask so that it accounted for 1% of the volume of themedium, and then cultured. The culture solution was centrifuged (6000×g,15 min, room temperature) and then 1 ml of the supernatant wasintroduced into a glass vial and thus designated as an analysis sample.

Ethyl Acetate Production Test

A transformant was cultured as follows. After active colony formation inan SD-His, Leu, Trp agar medium, cells were inoculated to 2 ml of anSD-His, Leu, Trp medium in a 15-ml test tube and then cultured overnightat 30 degrees C. (130 rpm). The thus pre-cultured solution wasinoculated to 100 ml of an SD-His, Leu, Trp medium in a 500-mlErlenmeyer flask so that it accounted for 1% thereof, and then cultured.The culture solution was centrifuged (6000×g, 15 min, room temperature)and then 1 ml of the supernatant was introduced into a glass vial sothat it was designated as an analysis sample.

Isopropanol Production Test

A transformant was cultured as follows. After active colony formation inan SD-His, Leu, Ura, Trp agar medium, cells were inoculated to 2 ml ofan SD-His, Leu, Ura, Trp medium in a 15-ml test tube and then culturedovernight at 30 degrees C. (130 rpm). The thus pre-cultured solution wasinoculated to 50 ml of an SD-His, Leu, Ura, Trp medium in a 500-mlErlenmeyer flask so that it accounted for 1% thereof, and then cultured.The culture solution was centrifuged (6000×g, 15 min, room temperature)and then 1 ml of the supernatant was introduced into a glass vial sothat it was designated as an analysis sample.

GC Analysis Conditions

Preparations used herein were acetic acid (NACALAI TESQUE, INC.), ethylacetate (NACALAI TESQUE, INC.), and isopropanol (NACALAI TESQUE, INC.).The following analytical instrument and analysis conditions wereemployed for the acetic acid production test, the ethyl acetateproduction test and the isopropanol production test.

TABLE 6 <Head space sampler analysis conditions> Head space samplerHP7694 (Hewlett-Packard) Zone Temp Oven 80° C. Loop 150° C. TR. LINE200° C. Event Time GC CYCLE TIME 10 min Vial EQ TIME 15 min PRESSURIZ.TIME 0.50 min Loop Fill TIME 0.2 min Loop EQ TIME 0.2 min INJECT TIME1.00 min Vial Prameter SHAKE HIGH Others Vial pressurization 15 psi Loopsize 3 ml <GC-MS analysis conditions> GC/MS HP6890/5973 GC/MS system(Hewlett-Packard, Wilmington, DE) Column HP-INNOWAX (Agilent:19091N-213) Inlet temperature 260° C. Detector temperature 260° C.Injection parameter Split ratio 1/20 Carrier gas Helium 1.0 ml/min Ovenheating conditions  60° C. 1 min Heat at 25° C./min to 260° C. 260° C. 1min

Yeast Metabolome Analysis by CE-TOFMS <Pre-Treatment>

Cells were cultured (30 degrees C.) in an SD-His, Leu, Ura, Trp mediumand then sampling was performed so that the amount of the sample was 15OD unit. Suction filtration of the resultant was immediately performedby filtration. Next, suction filtration was performed twice with 10 mLof Milli-Q water and then washing was performed. Yeast cells collectedon the filter were immersed in 2 mL of methanol containing internalreference material (5 μM) and then 1.6 mL of the resultant wastransferred into a centrifugation tube. 1600 μL of chloroform and 640 μLof Milli-Q water were added to the tube and then it was stirred,followed by centrifugation (2,300×g, 4 degrees C., 5 minutes). Aftercentrifugation, aqueous phase was transferred to 6 ultrafiltration tubes(250 μL each) (MILLIPORE, Ultrafree MC UFC3 LCC Centrifugal Filter Unit5 KDa). The tubes were centrifuged (9,100×g, 4 degrees C., 120 minutes)and thus ultrafiltration was performed. Each filtered fluid wassolidified to dryness, dissolved again in 50 μL of Milli-Q water, andthen subjected to measurement.

<Measurement>

In this test, anionic metabolite (anion mode) measurement was performedunder the following conditions (see references 1) to 3)).

-   Apparatus: Agilent CE-TOFMS system (Agilent Technologies)-   Capillary: Fused silica capillary i.d. 50 μm×80 cm-   Measurement conditions:-   Run buffer: Anion Buffer Solution (p/n: H3302-1021)-   Rinse buffer: Anion Buffer Solution (p/n: H3302-1022)-   Sample injection: Pressure injection 50 mbar, 25 sec-   CE voltage: Positive, 30 kV-   MS ionization: ESI Negative-   MS capillary voltage: 3,500 V-   MS scan range: m/z 50-1,000-   Sheath liquid: HMT Sheath Liquid (p/n: H3301-1020)

<References, Data>

-   1) T. Soga, D. N. Heiger: Amino acid analysis by capillary    electrophoresis electrospray ionization mass spectrometry. Anal.    Chem. 72: 1236-1241, 2000.-   2) T. Soga, Y. Ueno, H. Naraoka, Y. Ohashi, M. Tomita et al.:    Simultaneous determination of anionic intermediates for Bacillus    subtilis metabolic pathways by capillary electrophoresis    electrospray ionization mass spectrometry. Anal. Chem. 74:    2233-2239, 2002.-   3) T. Soga, Y. Ohashi, Y. Ueno, H. Naraoka, M. Tomita et al.:    Quantitative metabolome analysis using capillary electrophoresis    mass spectrometry. J. Proteome Res. 2: 488-494, 2003.-   4) http://vanted.ipk-gatersleben.de/

[Results] Acetic Acid Production Test

Yeast strains subjected to the acetic acid production test are listed inTable 7 and the test results are shown in FIG. 11.

TABLE 7 Composition Wild-type PFK1 PFK2 strain (Dis- (Dis- ZWF1 RPE1PKT(1) PKT(2) PKT(3) PKT(4) PKT(5) (YPH499) rupted) rupted) (Enhanced)(Enhanced) (Introduced) (Introduced) (Introduced) (Introduced)(Introduced) Control ◯ ◯ ◯ Sample 1 ◯ ◯ Sample 2 ◯ ◯ ◯ ◯ Sample 3 ◯ ◯ ◯◯ Sample 4 ◯ ◯ ◯ ◯ Sample 5 ◯ ◯ ◯ ◯ Sample 6 ◯ ◯ ◯ ◯ Sample 7 ◯ ◯ ◯Sample 8 ◯ ◯ ◯ ◯ ◯

In Table 7, PKT(1) denotes a Bifidobacterium animalis sub sp.lactis-derived phosphoketolase gene, PKT(2) denotes an Aspergillusoryzae RIB40-derived phosphoketolase I gene, PKT(3) denotes anAspergillus oryzae RIB40-derived phosphoketolase II gene, PKT(4) denotesa Nostoc punctiforme ATCC 29133-derived phosphoketolase gene, and PKT(5)denotes a Marinobacter aquaeolei ATCC 700491-derived phosphoketolasegene.

Based on the results shown in FIG. 11, it was revealed that attenuationof an endogenous phosphofructokinase gene of yeast as a host andintroduction of a phosphoketolase gene into the yeast can enhance theflux toward the pentose phosphate system rather than toward theglycolytic system (FIG. 2), so that acetic acid can be produced at ahigh level. It was also revealed that as a phosphoketolase gene, theBifidobacterium-derived gene, and particularly the Bifidobacteriumanimalis-derived gene, is excellent in acetic acid productivity. It wasrevealed based on the results that as phosphoketolase genes,phosphoketolase genes within the broken-line frame in the phylogenetictree shown in FIG. 3 are preferable in terms of acetic acidproductivity.

Furthermore, it was revealed based on the results shown in FIG. 11 thatwhen a phosphofructokinase gene is attenuated to weaken the flux towardthe glycolytic system, both the PFK1 gene and the PFK2 gene arepreferably disrupted. Moreover, it was revealed that through enhancementof enzyme genes involved in the pentose phosphate system (FIG. 2), theflux toward the pentose phosphate system (FIG. 2) can be furtherenhanced and acetic acid can be produced at an even higher level.

Ethyl Acetate Production Test

Yeast strains subjected to the ethyl acetate production test are listedin Table 8 and the test results are shown in FIG. 12.

TABLE 8 Composition Wild-type PFK1 strain (Dis- ZWF1 RPE1 PKT PTA(1)PTA(2) PTA(3) ACS1 ACS2 ATF1 (YPH499) rupted) (Enhanced) (Enhanced)(Introduced) (Introduced) (Introduced) (Introduced) (Enhanced)(Enhanced) (Enhanced) Control ◯ Sample 1 ◯ ◯ ◯ ◯ Sample 2 ◯ ◯ ◯ ◯ Sample3 ◯ ◯ ◯ ◯ Sample 4 ◯ ◯ ◯ ◯ Sample 5 ◯ ◯ ◯ ◯ Sample 6 ◯ ◯ ◯ ◯ ◯ ◯ Sample7 ◯ ◯ ◯ ◯ ◯ ◯ Sample 8 ◯ ◯ ◯ ◯ ◯ ◯ Sample 9 ◯ ◯ ◯ ◯ ◯ ◯ Sample 10 ◯ ◯ ◯◯ ◯ ◯

In Table 8, PTA(1) denotes a Bacillus subtilis sub sp. subtilisstr.168-derived phosphate acetyltransferase gene, PTA(2) denotes aBifidobacterium animalis sub sp. lactis AD011-derived phosphateacetyltransferase gene, and PTA(3) denotes an Escherichia coli K-12MG1655-derived phosphate acetyltransferase gene. In addition, in thistest, a Bifidobacterium animalis sub sp. lactis-derived phosphoketolasegene was used as a PKT gene.

As shown in FIG. 12, the control strain in which the ATF1 gene had beenintroduced into the wild-type strain (YPH499 strain) also exhibited moreimproved ethyl acetate productivity compared with that of the wild-typestrain. However, it was understood that through attenuation of anendogenous phosphofructokinase gene of yeast as a host and introductionof a phosphoketolase gene into the host, in addition to furtherintroduction of a phosphoacetyltransferase gene (PTA gene) orenhancement of an acetyl-CoA synthetase gene (ACS gene), ethyl acetateproductivity was further improved compared with that of the control. Itwas demonstrated based on the results and the metabolic overview mapshown in FIG. 4 that the amount of acetyl-CoA synthesized wassignificantly increased through attenuation of the endogenousphosphofructokinase gene and introduction of the phosphoketolase gene inaddition to further introduction of the phosphoacetyltransferase gene(PTA gene) or enhancement of the acetyl-CoA synthetase gene (ACS gene).Moreover, it was demonstrated that the thus synthesized acetyl-CoA isaccumulated extracellularly at a high level in the form of ethyl acetateby an ATF1 enzyme.

In particular, as a phosphoacetyltransferase gene to be introduced, aBacillus subtilis-derived gene is preferable in view of acetyl-CoAproductivity. Also, it was revealed that as an acetyl-CoA synthetasegene to be enhanced, the ACS1 gene is more preferable than the ACS2gene.

Furthermore, it was revealed based on the results shown in FIG. 12 thatwhen a phosphofructokinase gene is attenuated to weaken the flux towardthe glycolytic system, both the PFK1 gene and the PFK2 gene arepreferably disrupted. Moreover, it was revealed that through enhancementof enzyme genes involved in the pentose phosphate system (FIG. 2), theflux toward the pentose phosphate system (FIG. 2) can be increased moreand ethyl acetate can be produced at an even higher level.

Isopropanol Production Test

Yeast strains subjected to the isopropanol production test are listed inTable 9 and the test results are shown in FIG. 13.

TABLE 9 Composition Wild-type PFK1 ctfA, ctfB, strain (Dis- ZWF1 RPE1PKT PTA(1) PTA(2) PTA(3) ACS1 ACS2 adc, ipdh (YPH499) rupted) (Enhanced)(Enhanced) (Introduced) (Introduced) (Introduced) (Introduced)(Enhanced) (Enhanced) (Introduced) Control ◯ Sample 1 ◯ ◯ ◯ ◯ ◯ ◯ Sample2 ◯ ◯ ◯ ◯ ◯ ◯ Sample 3 ◯ ◯ ◯ ◯ ◯ ◯ Sample 4 ◯ ◯ ◯ ◯ ◯ ◯ Sample 5 ◯ ◯ ◯ ◯◯ ◯

In Table 9, PTA genes (1) to (3) denote the same genes as in Table 8.

As shown in FIG. 13, it was understood that the capacity to produceisopropanol can be imparted to yeast through introduction of a ctfAgene, a ctfB gene, an adc gene, and an ipdh gene into the wild-typestrain (YPH499 strain). It was also understood that isopropanolproductivity was significantly improved compared with that of thecontrol through attenuation of the endogenous phosphofructokinase geneand introduction of the phosphoketolase gene, in addition to furtherintroduction of the phosphoacetyltransferase gene (PTA gene) orenhancement of the acetyl-CoA synthetase gene (ACS gene).

It was demonstrated based on the results and the metabolic overview mapshown in FIG. 4 that the amount of acetyl-CoA synthesized wassignificantly increased through attenuation of the endogenousphosphofructokinase gene and introduction of the phosphoketolase gene,in addition to further introduction of the phosphoacetyltransferase gene(PTA gene) or enhancement of the acetyl-CoA synthetase gene (ACS gene).It was demonstrated that the thus synthesized acetyl-CoA is accumulatedextracellularly at a high level as isopropanol because of ctfA, ctfB,adc, and ipdh.

In particular, it was understood that as a phosphoacetyltransferase geneto be introduced, Bacillus subtilis-derived gene is preferable in viewof acetyl-CoA productivity. Furthermore, it was revealed that as anacetyl-CoA synthetase gene to be enhanced, the ACS1 gene is morepreferable than the ACS2 gene.

Metabolome Analysis of Yeast by CE-TOFMS

Metabolome analysis was conducted for: a wild-type strain (YPH499strain); a strain (denoted as YPH499ΔPFKα/ZWF-RPE) prepared byattenuating an endogenous phosphofructokinase gene of the YPH499 strainand enhancing an enzyme gene involved in the pentose phosphate system(FIG. 2); and a strain (denoted as YPH499ΔPFKα/ZWF-RPE, PKT-PTA)prepared by attenuating the endogenous phosphofructokinase gene of theYPH499 strain, enhancing an enzyme gene involved in the pentosephosphate system (FIG. 2), and introducing a phosphoketolase gene and aphosphoacetyltransferase gene. FIG. 14 shows the results of themetabolome analysis.

In FIG. 14, the amount of acetyl-CoA in the case of the YPH499 strain(wild-type strain) is found by adding up the amount resulting from theethanol-mediated acetyl-CoA synthetic pathway and the amount resultingfrom the intramitochondrial acetyl-CoA synthetic pathway. In addition,in yeast's acetyl-CoA synthetic pathway, these two pathways, theethanol-mediated acetyl-CoA synthetic pathway and the intramitochondrialacetyl-CoA synthetic pathway, are present.

Meanwhile, in the case of the YPH499ΔPFKα/ZWF-RPE strain, theethanol-mediated acetyl-CoA synthetic pathway becomes unfunctional. Thisis because NADP is consumed because of the presence of ZWF, and as aresult, ALD2 catalyzing the reaction for conversion of aldehyde toacetic acid becomes unfunctional (see acetic acid productivity of thecontrol strain in the acetic acid production test (FIG. 11)). Therefore,the amount of acetyl-CoA synthesized by the YPH499ΔPFKα/ZWF-RPE straincorresponds to the amount of acetyl-CoA generated via theintramitochondrial acetyl-CoA synthetic pathway. In addition,intramitochondrially synthesized acetyl-CoA is consumed withinmitochondria, and thus it cannot be used as a raw material forsubstances such as ethyl acetate and isopropanol. Therefore, unless theamount of acetyl-CoA to be synthesized in the cytosol of yeast cells isincreased, the amounts of substances to be synthesized using synthesizedacetyl-CoA cannot be increased.

The YPH499ΔPFKα/ZWF-RPE, PKT-PTA strain is a mutant strain prepared byintroducing the PKT gene and the PTA gene into the YPH499ΔPFKα/ZWF-RPEstrain, so as to construct a novel acetyl-CoA synthetic pathway (seeFIG. 2 and FIG. 4). Therefore, the amount of acetyl-CoA synthesized viathe above novel acetyl-CoA synthetic pathway can be evaluated bysubtracting the amount of acetyl-CoA synthesized by theYPH499ΔPFKα/ZWF-RPE strain from the amount of acetyl-CoA synthesized bythe YPH499ΔPFKα/ZWF-RPE, PKT-PTA strain. Specifically, it was concludedfrom the results shown in FIG. 14 that the amount of acetyl-CoAcorresponding to 48 pmol (found by 62−14=48 pmol) could be synthesizedvia the novel acetyl-CoA synthetic pathway.

[Reference Example]

In the reference example, preparation of the isopropanol-producing yeast(#15-10) used in the above examples is described.

Obtainment of Isopropanol Synthetic Gene

The following 4 genes were cloned from the genome of Clostridiumacetobutylicum ATCC824 strain to a pT7Blue vector.

-   adc (Acetoacetic acid decarboxylase)-   ctfA (Butyrate-acetoacetate CoA-transferase subunit A)-   ctfB (Butyrate-acetoacetate CoA-transferase subunit B)-   thiA (Acetyl-CoA acetyltransferase)    Construction of pT7Blue-ADC

PCR was performed under the following conditions.

Primers

(SEQ ID NO: 79) adc-F: 5′-ATGTTAAAGGATGAAGTAATTAAACAAATTAG-3′(SEQ ID NO: 80) adc-R: 5′-TTACTTAAGATAATCATATATAACTTCAGCTC-3′

Reaction Conditions

-   Template: 0.4 μg of Clostridium genomic DNA-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (5 minutes)−(95 degrees C. (30 seconds), 60    degrees C. (30 seconds), 72 degrees C. (2 minutes))×30 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

The thus amplified 735-bp fragment was blunt-end cloned to a pT7Bluevector using a Perfectly Blunt Cloning Kit (Novagen). The clonedsequence was sequenced, thereby confirming that it was the adc sequence(CA-P0165) of the Clostridium acetobutylicum ATCC824 strain. The thusobtained plasmid was designated as pT7Blue-ADC.

Construction of pT7Blue-CTFA

PCR was performed under the following conditions.

Primers

ctfA-F: (SEQ ID NO: 81) 5′-ATGAACTCTAAAATAATTAGATTTGAAAATTTAAGG-3′ctfA-R: (SEQ ID NO: 82) 5′-TTATGCAGGCTCCTTTACTATATAATTTA-3′

Reaction Conditions

-   Template: 0.4 μg of Clostridium genomic DNA-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (5 minutes)−(95 degrees C. (30 seconds), 60    degrees C. (30 seconds), 72 degrees C. (2 minutes)×30 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

The thus amplified 657-bp fragment was cloned to a Perfectly BluntCloning Kit (Novagen) in a similar manner. The cloned sequence wassequenced, thereby confirming that it was the ctfA sequence (CA-P0163)of the Clostridium acetobutylicum ATCC824 strain. The thus obtainedplasmid was designated as pT7Blue-CTFA.

Construction of pT7Blue-CTFB

PCR was performed under the following conditions.

Primers

(SEQ ID NO: 83) ctfB-F: 5′-ATGATTAATGATAAAAACCTAGCGAAAG-3′(SEQ ID NO: 84) ctfB-R: 5′-CTAAACAGCCATGGGTCTAAGTTC-3′

Reaction Conditions

-   Template: 0.4 μg of Clostridium genomic DNA-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (5 minutes)−(95 degrees C. (30 seconds), 60    degrees C. (30 seconds), 72 degrees C. (2 minutes))×30 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

The thus amplified 666-bp fragment was cloned using a Perfectly BluntCloning Kit (Novagen). The cloned sequence was sequenced, therebyconfirming that it was the ctfB sequence (CA-P0164) of the Clostridiumacetobutylicum ATCC824 strain. The thus obtained plasmid was designatedas pT7Blue-CTFB.

Construction of pDI626PGKpro

PCR was performed under the following conditions.

Primers

SacI-Ppgk1 FW: (SEQ ID NO: 85) 5′-TAGGGAGCTCCAAGAATTACTCGTGAGTAAGG-3′SacII-Ppgk1 RV: (SEQ ID NO: 86)5′-ATAACCGCGGTGTTTTATATTTGTTGTAAAAAGTAG-3′

Reaction Conditions

-   Template: 0.4 μg of yeast YPH499 genomic DNA-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (5 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (2 minutes))×25 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

After purification of the reaction solution using a MinElute PCRpurification kit (QIAGEN), the resultant was digested with restrictionenzymes Sac I and Sac II. Agarose gel electrophoresis was performed toexcise a 712-bp fragment, and then it was purified using a MinElute Gelextraction kit (QIAGEN). The resultant was ligated to a pDI626GAP (APP.Env. Micro., 2009, 5536) vector digested with restriction enzymes Sac Iand Sac II in a similar manner. The thus obtained sequence wassequenced, thereby confirming that a plasmid of interest had beenconstructed. The thus obtained plasmid was designated as pDI626PGKpro.

Construction of pDI626PGK

PCR was performed under the following conditions.

Primers

SalI-Tpgk1 FW: (SEQ ID NO: 87)5′-TTAAGTCGACATTGAATTGAATTGAAATCGATAGATC-3′ KpnI-Tpgk1 RV2:(SEQ ID NO: 88) 5′-TTAAGGTACCGCTTCAAGCTTACACAACAC-3′

Reaction Conditions

-   Template: 0.4 μg of the genomic DNA of yeast YPH499-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (5 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (2 minutes))×25 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

After purification of the reaction solution using a MinElute PCRpurification kit (QIAGEN), the resultant was digested with restrictionenzymes Sal I and Kpn I. Agarose gel electrophoresis was performed toexcise a 330-bp fragment, and then it was purified using a MinElute Gelextraction kit (QIAGEN). The resultant was ligated to a pDI626PGKprovector digested with restriction enzymes Sal I and Kpn I. The thusobtained sequence was sequenced, thereby confirming that a plasmid ofinterest had been constructed. The thus obtained plasmid was designatedas pDI626PGK.

Construction of pDI626PGK-T

pDI626PGK was digested with a restriction enzyme Sbf I, and then thereaction solution was purified using a MinElute PCR purification kit(QIAGEN). Subsequently, the resultant was blunt-ended using a Bluntingkit (TaKaRaBIO), and then further digested with a restriction enzyme KpnI. Agarose gel electrophoresis was performed to excise a 3650-bpfragment, and then it was purified using a MinElute Gel extraction kit(QIAGEN). Thus a vector for ligation thereof was constructed. Next,pRS524GAP (APP. Env. Micro., 2009, 5536) was digested with restrictionenzymes PmaC I and Kpn I. Agarose gel electrophoresis was performed toexcise a 765-bp fragment and then it was purified using a MinElute Gelextraction kit (QIAGEN), so as to prepare an insert. Ligation thereofwas performed. Joints of the thus obtained sequence were sequenced,thereby confirming that a plasmid of interest had been constructed. Thethus obtained plasmid was designated as pDI626PGK-T.

Construction of pCR2.1-iPDH

A DNA sequence optimized for Saccharomyces cerevisiae codons based onthe Clostridium beijerinckii NRRL B593-derived adh: NADP-dependentalcohol dehydrogenase gene sequence registered in the GenBank(http://www.neb.nih.gov/Genbank/index.html) was synthesized (Operon). Avector portion is pCR2.1 (Invitrogen). In addition, the synthesized DNAsequence is shown in SEQ ID NO: 89, and the amino acid sequence encodedby a coding region contained in the synthesized DNA sequence is shown inSEQ ID NO: 90. The plasmid was designated as pCR2.1-iPDH.

Construction of pDI626PGK-T-iPDH

pCR2.1-iPDH was digested with restriction enzymes Sac II and Sal I toexcise a 1080-bp fragment. The resultant was ligated to a pDI626PGK-Tvector digested with restriction enzymes Sac II and Sal I in a similarmanner. The obtained sequence was sequenced, thereby confirming that aplasmid of interest had been constructed. The thus obtained plasmid wasdesignated as pDI626PGK-T-iPDH.

Construction of pENT-ADC

PCR was performed using pDI626-ADC as a template and the followingprimers.

Primers

08-189-adc-attB1-Fw: (SEQ ID NO: 91)5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAGTTCGAGTTTATCATT ATC-3′08-189-adc-attB4-Rv: (SEQ ID NO: 92)5′-GGGGACAACTTTGTATAGAAAAGTTGGGTGGGCCGCAAATTAAAGCC TTC-3′

The thus obtained 1809-bp PCR product was introduced into a pDONR221P1-P4 donor vector by gateway BP reaction. The obtained clone wassequenced, thereby confirming that no mutation was present in any partof the nucleotide sequence of the insert. The thus obtained plasmid wasdesignated as pENT-ADC.

Construction of pENT-CTFA

PCR was performed using pDI626PGK-CTFA as a template and the followingprimers.

08-189-ctfA-attB4r-Fw: (SEQ ID NO: 93)5′-GGGGACAACTTTTCTATACAAAGTTGGCTTCAAGCTTACACAACACG G-3′08-189-ctfA-attB3r-Rv: (SEQ ID NO: 94)5′-GGGGACAACTTTATTATACAAAGTTGTCAAGAATTACTCGTGAGTAA GG-3′

The obtained 1823-bp PCR product was introduced into a pDONR221 P4r-P3rdonor vector by gateway BP reaction. The obtained clone was sequenced,thereby confirming that no mutation site was present in any part of thenucleotide sequence of the insert. The thus obtained plasmid wasdesignated as pENT-CTFA.

Construction of pDI626-CTFB

pT7Blue-CTFB was digested with restriction enzymes BamH I and Sal I toexcise a 771-bp fragment. The resultant was ligated to a pDI626 vectordigested with restriction enzymes BamH I and Sal I in a similar manner.The obtained sequence was sequenced, thereby confirming that a plasmidof interest had been constructed. The thus obtained plasmid wasdesignated as pDI626-CTFB(+A).

PCR was performed under the following conditions using the followingprimers in order to correct mutation sites in primers.

BamHI-ctfB-F: (SEQ ID NO: 95) 5′-TAGTGGATCCGATGATTAATGATAAAAACC-3′pDI626MCS-seqF: (SEQ ID NO: 96) 5′-CCTAGACTTCAGGTTGTCTAAC-3′

Reaction Conditions

-   Template: 1 ng of pDI626-CTFB(+A)-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl of Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (2 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (1 minutes)×20 cycles−72    degrees C. (3 minutes)−4 degrees C. (stock)

After purification of the reaction solution using a MinElute PCRpurification kit (QIAGEN), the resultant was digested with restrictionenzymes BamH I and Sal I. Agarose gel electrophoresis was performed toexcise a 702-bp fragment and then it was purified using a MinElute Gelextraction kit (QIAGEN). The resultant was ligated to a pDI626 vectordigested with restriction enzymes BamH I and Sal I. The thus obtainedsequence was sequenced, thereby confirming that mutation sites had beencorrected. The thus obtained plasmid was designated as pDI626-CTFB.

Construction of pENT-CTFB

PCR was performed using pDI626-CTFB as a template and the followingprimers.

08-189-ctfB-attB3-Fw: (SEQ ID NO: 97)5′-GGGGACAACTTTGTATAATAAAGTTGGGCCGCAAATTAAAGCCTTC- 3′08-189-ctfB-attB2-Rv: (SEQ ID NO: 98)5′-GGGGACCACTTTGTACAAGAAAGCTGGGTACAGTTCGAGTTTATCAT TATC-3′

The thus obtained 1737-bp PCR product was introduced into a pDONR221P3-P2 donor vector by gateway BP reaction. The obtained clone wassequenced, thereby confirming that no mutation site was present in anypart of the nucleotide sequence of the insert. The thus obtained plasmidwas designated as pENT-CTFB.

Construction of pDEST626(2008)

PCR was performed under the following conditions.

Primers

SacI-convA-F: (SEQ ID NO: 99) 5′-TAGGGAGCTCATCACAAGTTTGTACAAAAAAGCTG-3′KpnI-convA-R: (SEQ ID NO: 100) 5′-TTAAGGTACCATCACCACTTTGTACAAGAAAGC-3′

Reaction Conditions

-   Template: 0.5 ng of RfA (Invitrogen; Reading Frame Cassette A of    Gateway Vector Conversion System)-   Primer: 50 pmol primer DNA-   Reaction solution: 50 μl of the solution containing 1× Pfu Ultra II    reaction buffer (Stratagene), 10 nmol dNTP, and 1 μl Pfu Ultra II    fusion HS DNA polymerase (Stratagene)-   Reaction: 95 degrees C. (2 minutes)−(95 degrees C. (30 seconds), 55    degrees C. (30 seconds), 72 degrees C. (1 minute and 30 seconds)×20    cycles−72 degrees C. (3 minutes)−4 degrees C. (stock)

After purification of the reaction solution using a MinElute PCRpurification kit (QIAGEN), the resultant was digested with restrictionenzymes Sac I and Kpn I. Agarose gel electrophoresis was performed toexcise a 1717-bp fragment. After purification using a MinElute Gelextraction kit (QIAGEN), the resultant was ligated to the pDI626GAPvector (APP. Env. Micro., 2009, 5536) digested with restriction enzymesSac I and Kpn I. The obtained sequence was sequenced, thereby confirmingthat a plasmid of interest had been constructed.

Construction of pEXP(Ura)-ADC-CTFA-CTFB

The 3 obtained entry clones (pENT-ADC, pENT-CTFA, and pENT-CTFB) wereincorporated into a pDEST626(2008) expression vector by Gateway LRreaction. The thus obtained clones were confirmed by PCR for insertsize, thereby confirming correct recombination. Sequencing wasperformed, thereby confirming that no error was present in the sequence.The thus obtained plasmid was designated as pEXP(Ura)-ADC-CTFA-CTFB.

Preparation of #3-17 Strain

The pEXP(Ura)-ADC-CTFA-CTFB expression vector was cleaved and linearizedwith restriction enzymes Aat II and BssH II. After ethanolprecipitation, the resultant was dissolved in 0.1× TE Buffer and thenSaccharomyces cerevisiae YPH499 (Stratagene) was transformed using aFrozen EZ yeast transformation kit (Zymoresearch). The obtained cloneswere subjected to colony PCR, thereby confirming 25 clones into whichadc, ctfA, and ctfB genes had been introduced. The strain with thehighest acetone production amount was designated as #3-17.

Preparation of #15-10 Strain

The pDI626PGK-T-iPDH expression vector of the ipdh gene that was asynthetic gene expected to convert acetone to isopropanol was cleavedand linearized with restriction enzymes Aat II and BssH II. Afterethanol precipitation, the resultant was dissolved in 0.1× TE Buffer,and then the #3-17 acetone-producing yeast was transformed using aFrozen EZ yeast transformation kit (Zymoresearch). The thus obtained 14clones were subjected to colony PCR, thereby confirming 13 clones inwhich the ipdh gene had been introduced. The strain with the highestisopropanol production amount was designated as #15-10.

1. A recombinant, non-xylose fermenting Saccharomyces cerevisiae, whichcomprises an attenuated phosphofructokinase gene endogenous to saidSaccharomyces cerevisiae, an introduced phosphoketolase gene, anintroduced Clostridium acetobutylicum acetoacetic acid decarboxylasegene, an introduced Clostridium acetobutylicum butyrate-acetoacetateCoA-transferase subunit A gene, an introduced Clostridium acetobutylicumbutyrate-acetoacetate CoA-transferase subunit B gene, an introducedClostridium acetobutylicum acetyl-CoA acetyltransferase gene, and anintroduced Clostridium acetobutylicum isopropanol dehydrogenase gene,wherein isopropanol production by said Saccharomyces cerevisiae isenhanced compared with wild-type strain, and wherein said introducedphosphoketolase gene encodes the amino acid sequence of any one of SEQID NOS: 1 to
 19. 2. A method for producing isopropanol, comprising astep of culturing the recombinant yeast of claim 1 in medium.